Project Summary There is an obvious need for new approaches and better assays for the laboratory diagnosis of Lyme disease. All attempts to develop a practical assay for the direct detection of Borrelia burgdorferi in infected patients have failed. Thus far, all proposed alternatives to serology have been unsuccessful, not cost-effective, or are still in early development. Improving serological detection in early disease is the fastest and most effective way to improve patient outcomes in Lyme disease. The antigen targets utilized in current serodiagnostic assays have considerable defects. They often contain conserved epitopes that cross-react with antibodies raised to other antigens, reducing specificity and requiring the use of a two-tier seroassay paradigm that preserves specificity at the cost of poor sensitivity in the detection of early disease. The use of peptides as serodiagnostic targets demonstrate improved efficacy, but the use of one or two peptides containing single epitopes still provides re- duced sensitivity in early disease. Putting the same old antigen targets into new platforms, no matter how in- novative the platform, will not succeed in improving serodiagnostics for Lyme disease. Both the target anti- gens and assays need an innovative approach. By incorporating unique peptides containing linear epitopes highly specific to B. burgdorferi, into a cutting-edge, multiplex, portable paper-based point-of-care diagnostic assay that uses a cost-effective smartphone-based reader, we aim to transform the diagnosis of Lyme disease. An ideal test for Lyme disease could be performed in a single step and yield an answer on the spot to support diagnosis and direct the course of clinical treatment. We have developed an innovative vertical-flow assay which allows for the multiplexed detection of IgM and IgG binding of up to 25 independent antigen targets in a point-of-care setting. The VFA design allows for uniform flow of sample across the target membrane, creating uniform binding conditions and maximizing developed signal. We coupled this design with a cost-effective portable smart-phone based reader allowing for quantitative measure of antibody binding, eliminating subjectiv- ity. By multiplexing peptide antigens each containing 1-2 epitopes unique to B. burgdorferi from multiple differ- ent antigens expressed at different stages during mammalian infection we can generate a single tier, POC as- say that can specifically and sensitively detect patient antibody at all stages of the disease. The assay can be completed in less than 25 min allowing for rapid in office results to support clinical diagnosis, thereby improving patient outcomes.